Fusion proteins for prodrug activation

ABSTRACT

The invention relates to compounds which contain an antigen binding region which is bound to at least one enzyme which is able to metabolize a compound (prodrug) which has little or no cytotoxicity to a cytotoxic compound (drug), where the antigen binding region is composed of a single polypeptide chain. It is advantageous for covalently bonded carbohydrates to be present on the polypeptide chain.

RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.08/989,896 filed on Dec. 12, 1997, now U.S. Pat. No. 7,060,495, issuedJun. 13, 2006, which is a continuation of U.S. application Ser. No.08/475,826, filed on Jun. 7, 1995 now abandoned which is a divisional ofU.S. application Ser. No. 08/404,949 filed Mar. 15, 1995 now abandoned,which is a continuation of U.S. application Ser. No. 08/129,379 filed onSep. 30, 1993 now abandoned and claims priority from German patentapplication P 42 33 152.8 filed Oct. 2, 1992.

The invention relates to compounds which contain an antigen bindingregion which is bound to at least one enzyme which is able to metabolizea compound (prodrug) which has little or no cytotoxicity to a cytotoxiccompound (drug), where the antigen binding region is composed of asingle polypeptide chain. It is advantageous for covalently bondedcarbohydrates to be present on the polypeptide chain.

The combination of prodrug and antibody-enzyme conjugates for use astherapeutic composition has already been described in the specialistliterature. This entails antibodies which are directed against aparticular tissue and to which a prodrug-cleaving enzyme is bound beinginjected into an organism, and subsequently a prodrug compound which canbe activated by the enzyme being administered. The action of theantibody-enzyme conjugate bound to the target tissue is intended toconvert the prodrug compound into a compound which exerts a cytotoxiceffect on the bound tissue. However, studies on antibody-enzymeconjugates have shown that these chemical conjugates have unfavorablepharmacokinetics so that there is only inadequate site-specifictumor-selective cleavage of the prodrug. Some authors have attempted toremedy this evident deficiency by additional injection of an anti-enzymeantibody which is intended to bring about rapid elimination of theantibody-enzyme conjugate from the plasma (Sharma et al., Brit. J.Cancer, 61, 659, 1990). Another problem of antibody-enzyme conjugates isthe limited possibility of preparing large amounts reproducibly andhomogeneously.

The object of the present invention was now to find fusion proteinswhich can be prepared on an industrial scale and are suitable, by reasonof their pharmacokinetic and pharmacodynamic properties, for therapeuticuses.

It has been found in this connection that compounds which contain anantigen binding region which is composed of a single polypeptide chainhave unexpected advantages for the preparation and use of fusionproteins, to which carbohydrates are advantageously attached, in prodrugactivation.

The invention therefore relates to compounds which contain an antigenbinding region which is bound to at least one enzyme, where the antigenbinding region is composed of a single polypeptide chain, andcarbohydrates are advantageously attached to the fusion protein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates the purification of the sFv-huβ-Gluc fusion proteinby TSK 3000 gel chromatography.

FIG. 2 shows the nucleotide sequences of oligonucleotides pAB-Back,linker-anti, linker-sense, and V_(L(Mut))-For.

FIG. 3 is a schematic representation of the amplification of the V_(H)gene, including the signal sequence intrinsic to the V_(H) gene, fromthe plasmid pABstop 431V_(H)hum (V_(H)431/26) by PCR usingoligonucleotides pAB-Back and linker-anti, and the amplification of theV_(L) gene from pABstop 431V_(L)hum (V_(L)431/26) by PCR usingoligonucleotides linker-sense and V_(L(Mut))-For.

FIG. 4 is a schematic representation of the amplification and fusion ofthe V_(H)431/26 and the V_(L)431/26 gene fragments by PCR.

FIG. 5 is a schematic representation of the cloning of the sFv 431/26fragment into the expression vector Pab 431V_(H)hum/C_(H)1+1H/β-Glc,which contains the huβ-glucuronidase gene.

FIG. 6 is a schematic representation of the plasmid pRHM 140, whichharbors a neomycin-resistance gene.

FIG. 7 is a schematic representation of the plasmid pSV2, which harborsa methotrexate-resistance gene.

FIG. 8 shows the nucleotide sequence of oligonucleotides sFv for (2561),sFv back (2577), Hum.β-Gluc. back oligo (2562), Hum.β-Gluc. for oligo(2540), PCR oligo VHpIXY back (2587), and PCR oligo VKpIXY for (2627).

FIG. 9 is a schematic representation of the amplification of thesingle-chain Fv, sFv 431/26, by PCR using oligonucleotides 2561 and2577, and the cloning of that single-chain Fv into the vector pUC19.

FIG. 10 is a schematic representation of the amplification of the humanβ-glucuronidase gene from the plasmid pAB 431 V_(H)hum/CH1+1H/huβ-Glucby PCR using oligonucleotides 2562 and 2540, and the ligation of thatgene into the plasmid sFv 431/26 in pUC19.

FIG. 11 is a schematic representation of the amplification of aKpnI/NcoI fragment from the sFv 431/26 by PCR using oligonucleotides2587 and 2627, and the cloning of that fragment into the yeastexpression vector pIXY.

FIG. 12 is a schematic representation of the ligation the BstEII/HindIIIfragment from the plasmid sFv 431/26 huβ-Gluc in pUC19 into the vectorpIXY 120 containing a V_(H) gene, a linker, and a part of a V_(L) gene.

FIG. 13 shows the nucleotide sequence of oligonucleotides E. coliβ-Gluc. for (2639) and E. coli β-Gluc. back (2638).

FIG. 14 is a schematic representation of the amplification of the E.coli glucuronidase gene from the plasmid pRAJ275 by PCR usingoligonucleotides 2638 and 2639, and the ligation of that gene into sFv431/26 in pUC19.

FIG. 15 is a schematic representation of the cloning of theBstEII/HindIII fragment from the plasmid sFv 431/26 E. coli β-Gluc inpUC19 into the vextor pIXY 120.

FIG. 16 shows the nucleotide sequences of oligonucleotides PCR oligoVHpIXY back (2587), PCR oligo VKpIXY/β-lactamase for (2669), PCR oligolink/β-lactamase back (2673), and PCR oligo β-lactamase for (2764).

FIG. 17 is a schematic representation of the amplification of sFv 431/26by PCR using oligonucleotides 2587 and 2669, and the cloning of sFv431/26 into the vector pUC19.

FIG. 18 is a schematic representation of the amplification of theβ-lactamase II gene from the complete DNA of Bacillus cereus by PCRusing oligonucleotides 2673 and 2674, and the cloning of that gene intothe vector pUC19.

FIG. 19 is a schematic representation of the ligation of a BclI/HindIIIfragment of the β-lactamase gene into sFv 431/26 in pUC19.

FIG. 20 is a schematic representation of the ligation of theKpnI/HindIII sFv β-lactamase fragment into the vector pIXY 120.

An antigen binding region means for the purpose of the invention aregion which contains at least two variable domains of an antibody,preferably one variable domain of a heavy antibody chain and onevariable domain of a light antibody chain (sFv fragment). The antigenbinding region can, however, also have a bi- or multivalent structure,i.e. two or more binding regions, as described, for example, in EP-A-0404 097. However, a human or humanized sFv fragment is particularlypreferred, especially a humanized sFv fragment.

The antigen binding region preferably binds to a tumor-associatedantigen (TAA), with the following TAAs being particularly preferred:

-   neural cell adhesion molecule (N-CAM),-   polymorphic epithelial mucin (PEM),-   epidermal growth factor receptor (EGF-R),-   Thomsen Friedenreich antigen β (TFβ),-   gastrointestinal tract carcinoma antigen (GICA),-   ganglioside GD₃ (GD3),-   ganglioside GD₂ (GD₂),-   Sialyl-Le^(a), Sialyl-Le^(x),-   TAG72,-   the 24-25 kDa glycoprotein defined by MAb L6,-   CA 125 and, especially,-   carcinoembryonic antigen (CEA).

Preferred enzymes are those enzymes which are able to metabolize acompound of little or no cytotoxicity to a cytotoxic compound. Examplesare β-lactamase, pyroglutamate aminopeptidase, galactosidase orD-aminopeptidase as described, for example, in EP-A2-0 382 411 orEP-A2-0 392 745, an oxidase such as, for example, ethanol oxidase,galactose oxidase, D-amino-acid oxidase or α-glyceryl-phosphate oxidaseas described, for example, in WO 91/00108, peroxidase as disclosed, forexample, in EP-A2-0 361 908, a phosphatase as described, for example, inEP-A1-0 302 473, a hydroxynitrilelyase or glucosidase as disclosed, forexample, in WO 91/11201, a carboxypeptidase such as, for example,carboxypeptidase G2 (WO 88/07378), an amidase such as, for example,penicillin 5-amidase (Kerr, D. E. et al. Cancer Immunol. Immunther.1990, 31) and a protease, esterase or glycosidase such as the alreadymentioned galactosidase, glucosidase or a glucuronidase as described,for example, in WO 91/08770.

A β-glucuronidase is preferred, preferably from Kobayasia nipponica orSecale cereale, and more preferably from E. coli or a humanβ-glucuronidase. The substrates for the individual enzymes are alsoindicated in the said patents and are intended also to form part of thedisclosure content of the present application. Preferred substrates ofβ-glucuronidase are N-(D-glycopyranosyl)benzyloxycarbonylanthracyclinesand, in particular, N-(4-hydroxy3-nitrobenzyloxycarbonyl)doxorubicin anddaunorubicin β-D-glucuronide (J. C. Florent et al. (1992) Int.Carbohydr. Symp. Paris, A262, 297 or S. Andrianomenjanahary et al.(1992) Int. Carbohydr. Symp. Paris, A 264, 299).

The invention further relates to nucleic acids which code for thecompounds according to the invention. Particularly preferred is anucleic acid, as well as its variants and mutants, which codes for ahumanized sFv fragment against CEA (carcinoembryonic antigen) linked toa human β-glucuronidase, preferably with the nucleotide sequence of SEQID NO:1, which codes for the amino acid sequence of SEQ ID NO:2.

The compounds according to the invention are prepared in general bymethods of genetic manipulation which are generally known to the skilledworker, it being possible for the antigen binding region to be linked toone or more enzymes either directly or via a linker, preferably apeptide linker. The peptide linker which can be used is, for example, ahinge region of an antibody or a hinge-like amino-acid sequence. In thiscase, the enzyme is preferably linked with the N terminus to the antigenbinding region directly or via a peptide linker. The enzyme or enzymescan, however, also be linked to the antigen binding region chemically asdescribed, for example, in WO 91/00108.

The nucleic acid coding for the amino-acid sequence of the compoundsaccording to the invention is generally cloned in an expression vector,introduced into prokaryotic or eukaryotic host cells such as, forexample, BHK, CHO, COS, HeLa, insect, tobacco plant, yeast or E. colicells and expressed. The compound prepared in this way can subsequentlybe isolated and used as diagnostic aid or therapeutic agent. Anothergenerally known method for the preparation of the compound according tothe invention is the expression of the nucleic acids which code thereforin transgenic mammals with the exception of humans, preferably in atransgenic goat.

BHK cells transfected with the nucleic acids according to the inventionexpress a fusion protein (sFv-huβ-Gluc) which not only was specific forCEA but also had full β-glucuronidase activity (see Example 5).

This fusion protein was purified by anti-idiotype affinitychromatography in accordance with the method described in EP 0 501 215A2 (Example M). The fusion protein purified in this way gives amolecular weight of 100 kDA in the SDS PAGE under reducing conditions,while molecules of 100 and 200 kDa respectively appear undernon-reducing conditions.

Gel chromatography under native conditions (TSK-3000 gel chromatography)showed one protein peak (Example 6, FIG. 1) which correlates with theactivity peak in the specificity enzyme activity test (EP 0 501 215 A2).The position of the peak by comparison with standard molecular weightmarkers indicates a molecular weight of ≈200 kDa. This finding, togetherwith the data from the SDS PAGE, suggests that the functionalenzymatically active sFv-huβ-Gluc fusion protein is in the form of a“bivalent molecule”, i.e. with 2 binding regions and 2 enzyme molecules.Experiments not described here indicate that the fusion protein may,under certain cultivation conditions, be in the form of a tetramer with4 binding regions and 4 enzyme molecules. After the sFv-huβ-Gluc fusionprotein had been purified and undergone functional characterization invitro, the pharmacokinetics and the tumor localization of the fusionprotein were determined in nude mice provided with human gastriccarcinomas. The amounts of functionally active fusion protein weredetermined in the organs and in the tumor at various times afterappropriate workup of the organs (Example 7) and by immunologicaldetermination (triple determinant test, Example 8). The results of arepresentative experiment are compiled in Table 1.

Astonishingly, a tumor/plasma ratio of 5/1 is reached after only 48hours. At later times, this ratio becomes even more favorable andreaches values >200/1 (day 5). The reason for this favorablepharmacokinetic behavior of the sFv-huβ-Gluc fusion protein is thatfusion protein not bound to the tumor is removed from the plasma and thenormal tissues by internalization mainly by receptors for mannose6-phosphate and galactose. (Evidence for this statement is that there isan intracellular increase in the β-glucuronidase level, for example inthe liver).

As shown in Table 2, the sFv-huβ-Gluc contains relatively large amountsof galactose and, especialty, mannose, which are mainly responsible forthe binding to the particular receptors. The fusion protein/receptorcomplex which results and in which there is binding via the carbohydrateresidues of the fusion protein is then removed from the extracellularcompartment by internalization.

This rapid internalization mechanism, which is mainly mediated bygalactose and mannose, is closely involved in the advantageousphannacokinetics of the fusion protein according to the invention. Theseadvantageous pharmacokinetics of the fusion protein to which galactoseand, in particular, mannose are attached makes it possible for ahydrophilic prodrug which undergoes extracellular distribution to beadministered i.v. at a relatively early time without elicitingnon-specific prodrug activation. In this case an elimination step asdescribed by Sharma et al. (Brit. J. Cancer, 61, 659, 1990) isunnecessary. Based on the data in Table 1, injection of a suitableprodrug (S. Adrianomenjanahari et al. 1992, Int. Carbohydrate Symp.,Parts A264, 299) is possible even 3 days after injection of thesFv-huβ-Gluc fusion protein without producing significant side effects(data not shown).

A similarly advantageous attachment of carbohydrates to fusion proteinscan also be achieved, for example, by secretory expression of thesFv-huβ-Gluc fusion protein in particular yeast strains such asSaccharomyces cerevisiac or Hansenula polymorpha. These organisms arecapable of very effective mannosylation of fusion proteins which haveappropriate N-glycosylation sites (Goochee et al., Biotechnology, 9,1347-1354, 1991). Such fusion proteins which have undergone secretoryexpression in yeast cells show a high degree of mannosylation andfavorable pharmacokinetics comparable to those of the sFv-huβ-Glucfusion protein expressed in BHK cells (data not shown). In this case,the absence of galactose is compensated by the even higher degree ofmannosylation of the fusion protein Table 3). The sFv-huβ-Gluc fusionprotein described above was constructed by genetic manipulation andexpressed in yeast as described in detail in Example 9.

Instead of human β-glucuronidase it is, however, also possible to employanother glucuronidase with advantageous properties. For example, the E.coli β-glucuronidase has the particular advantage that its catalyticactivity at pH 7.4 is signiflcantly higher than that of humanβ-glucuronidase. In Example 10, an sFv-E. coli β-Gluc construct wasprepared by methods of genetic manipulation and underwent secretoryexpression as functionally active mannosylated fusion protein inSaccharomyces cerevisiae. The pharmacokinetic data are comparable tothose of the sFv-huβ-Gluc molecule which was expressed in yeast or inBHK cells (Table 1).

The glucuronidases from the fungus Kobayasia nipponica and from theplants Secale cereale have the advantage, for example, that they arealso active as monomers. In Example 11, methods of genetic manipulationwere used to prepare a construct which, after expression inSaccharomyces cerevisiae, excretes an sFv-β. cereus β-lactamase IIfusion protein preferentially in mannosylated form.

This fusion protein likewise has, as the fusion proteins according tothe invention, on the basis of β-glucuronidase pharmacokinetics whichare favorable for prodrug activation (Table1).

Furthermore, the compounds according to the invention can be employednot only in combination with a prodrug but also in the framework ofconventional chemotherapy in which cytostatics which are metabolized asglucuronides and thus inactivated can be converted back into their toxicform by the administered compounds.

The following examples now describe the synthesis by geneticmanipulation of sFv-β-Gluc fusion proteins, and the demonstration of theability to function.

The starting material comprised the plasmids pABstop 431/26 hum V_(H)and pABstop 431/26 hum VH_(L). These plasmids contain the humanizedversion of the V_(H) gene and V_(L) gene of anti-CEA MAb BW 431/26(Gussow and Seemann, 1991, Meth. Enzymology, 203, 99-121). Furtherstarting material comprised the plasmid pABstop 431/26 V_(H)-huβ-Gluc 1H(EP-A2-0 501 215) which contains a V_(H) exon, including theV_(H)-intrinsic signal sequence, followed by a CH1 exon, by the hingeexon of a human IgG3 C gene and the complete cDNA of humanβ-glucuronidase.

EXAMPLE 1

Amplification of the V_(H) and V_(L) Genes of MAb hum 431/26

The oligonucleotides pAB-Back (SEQ ID NO:3) and linker-anti (SEQ IDNO:4) are used to amplify the V_(H) gene including the signal sequenceintrinsic to the V_(H) gene from pABstop 431V_(H) hum (V_(H) 431/26)(FIG. 3) (Gussox and Seemann, 1991, Meth. Enzymology, 203, 99-121). Theoligonucleotides linker-sense (SEQ ID NO:5) and V_(L)(Mut)-For (SEQ IDNO:6)(FIG. 2) are used to amplify the V_(L) gene from pABstop 431V_(L)hum (V_(L) 431/26) (FIG. 3).

EXAMPLE 2

Joining of the V_(H) 431/26 and V_(L) 431/26 Gene Fragments

The oligonucleotides linker-anti and linker-sense are partiallycomplementary with one another and encode a polypeptide linker which isintended to link the V_(H) domain and V_(L) domain to give an sFvfragment. In order to fuse the amplified V_(H) fragments with the V_(L)fragments, they are purified and employed in a 10-cycle reaction asfollows:

H₂O: 37.5 μl dNTPs (2.5 mM): 5.0 μl PCR buffer (10×): 5.0 μl Taqpolymerase (Perkin-Elmer Corp., Emmeryville, CA) 0.5 μl (2.5 U/μl): 0.5μg/μl DNA of the V_(L) frag.: 1.0 μl 0.5 μg/μl DNA of the V_(H) frag.:1.0 μl PCR buffer (10×): 100 mM tris, pH 8.3, 500 mM KCl, 15 mM MgCl2,0.1% (w/v) gelatin.

The surface of the reaction mixture is sealed with paraffin, andsubsequently the 10-cycle reaction is carried out in a PCR apparatusprogrammed for 94° C., 1 min; 55° C., 1 min; 72° C., 2 min. 2.5 pmol ofthe flanking primer pAB-Back and V_(L)(Mut)-For are added, and a further20 cycles are carried out. The resulting PCR fragment is composed of theV_(H) gene which is linked to the V_(L) gene via a linker (FIG. 4). Thesignal sequence intrinsic to the V_(H) gene is also present in front ofthe V_(H) gene.

The oligonucleotide V_(L)(Mut)-For also results in the last nucleotidebase of the V_(L) gene, a C, being replaced by a G. This PCR fragmentcodes for a humanized single-chain Fv (sFV 431/26).

EXAMPLE 3

Cloning of the sFv 431/26 Fragment into the Expression Vector whichContains the huβ-Glucuronidase Gene.

The sFv fragment from (2) is cut with HindIII and BamHI and ligated intothe vector pAB 431V_(H)hum/CH1+1h/β-Glc which has been completelycleaved with HindIII and partially cleaved with BglII. The vectorpABstop 431/26V_(H)huβ-Gluc1H contains a V_(H) exon, including theV_(H)-intrinsic signal sequence, followed by a CH1 exon, by the hingeexon of a human IgG3 C gene and by the complete cDNA of humanβ-glucuronidase. The plasmid clone pMCG-E1 which contains the humanizedsFv 431/26, a hinge exon and the gene for human β-glucuronidase isisolated (pMCG-E1)(FIG. 5).

EXAMPLE 4

Expression of the sFv-huβ-Gluc Fusion Protein in BHK Cells.

The clone pMCG-E1 is transfected with the plasmid pRMH 140 which harborsa neomycin-resistance gene (FIG. 6) and with the plasmid pSV2 whichharbors a methotrexateresistance gene (FIG. 7) into BHK cells. The BHKcells subsequently express a fusion protein which has both theantigen-binding properties of MAb BW 431/26hum and the enzymaticactyvity of human β-glucuronidase. 4

EXAMPLE 5

Demonstration of the Antigen-Binding Properties and of the EnzymaticActivity of the sFv-huβ-Gluc Fusion Protein.

The ability of the sFv-huβ-Gluc fusion protein to bind specifically tothe CEA epitope defined by 431/26 and simultaneously to exert theenzymatic activity of human β-glucuronidase was shown in a specificityenzyme activity test (EP-A2-0 501 215). The test determines theliberation of 4-methylumbelliferone from 4-methylumbelliferylβ-glucuronide by the β-glucuronidase portion of the fusion protein afterthe fusion protein has been bound via the sFv portion to an antigen. Themeasured fluorescence values are reported as relative fluorescence units(FU). The test shows a significant liberation of methyl-umbelliferone bythe fusion protein in the plates coated with CEA. By contrast, thefusion protein does not liberate any methylumbelliferone in controlplates coated with PEM (polymorphic epithelial mucin).

EXAMPLE 6

TSK 3000 Gel Chromatography

200 ng of the sFv-huβ-Gluc fusion protein which had been purified byanti-idiotype affinity chromatography in 25 μl were chromatographed on aTSK gel G 3000 SW XL column (TOSO HAAS Order No. 3.5Wx.N3211, 7.8 mm×300mm) in a suitable mobile phase (PBS, pH 7.2, containing 5 g/l maltoseand 4.2 g/l arginine) at a flow rate of 0.5 ml/min. The Merck HitachiHPLC system (L-4000 UV detector, L-6210 intelligent pump, D-2500Chromato-integrator) was operated under ≈20 bar, the optical density ofthe eluate was determined at 280 nm, and an LKB 2111 Multisac fractioncollector was used to collect 0.5 ml fractions which were subsequentlyanalysed in a specificity enzyme activity test (SEAT) (EP 0 501 215 A2,Example J). The result of this experiment is shown in FIG. 1. It isclearly evident that the position of the peak detectable by measurementof the optical density at 280 nm coincides with the peak whichdetermines the specificity and enzyme activity (SEAT) of the eluate.Based on the positions of the molecular weights of standard proteinswhich are indicated by arrows, it can be concluded that the functionallyactive sFv-huβ-Gluc fusion protein has an approximate molecular weightof ≈200 kDa under native conditions.

EXAMPLE 7

Workup of Organs/Tumors for Determination of the Fusion Protein

The following sequential steps were carried out:

-   nude mice (CD1) which have a subcutaneous tumor and have been    treated with fusion protein or antibody-enzyme conjugate undergo    retroorbital exsanguination and are then sacrificed-   the blood is immediately placed in an Eppendorf tube which already    contains 10 μl of Liquemin 25000 (from Hoffman-LaRoche AG)-   centrifugation is then carried out in a centrifuge (Megafuge 1.0,    from Heraeus) at 2500 rpm for 10 min-   the plasma is then obtained and frozen until tested-   the organs or the tumor are removed and weighed-   they are then completely homogenized with 2 ml of 1% BSA in PBS, pH    7.2-   the tumor homogenates are adjusted to pH 4.2 with 0.1 N HCl (the    sample must not be overtitrated because β-glucuronidase is    inactivated at pH<3.8)-   all the homogenates are centrifuged at 16000 g for 30 min-   the clear supernatant is removed-   the tumor supernatants are neutralized with 0.1 N NaOH-   the supernatants and the plasma can now be quantified in    immunological tests.

EXAMPLE 8

Triple Determinant Test

The tests are carried out as follows:

-   75 μl of a mouse anti-huβ-Gluc antibody (MAb 2118/157 Behringwerke)    diluted to 2 μg/ml in PBS, pH 7.2, are placed in each well of a    microtiter plate (polystyrene U-shape, type B, from Nunc, Order No.    4-60445)-   the microtiter plates are covered and incubated at R.T. overnight-   the microtiter plates are subsequently washed 3× with 250 μl of 0.05    M tris-citrate buffer, pH 7.4, per well-   these microtiter plates coated in this way are incubated with 250 μl    of blocking solution (1% casein in PBS, pH 7.2) in each well at R.T.    for 30′ (blocking of non-specific binding sites) (coated microtiter    plates which are not required are dried at R.T. for 24 hours and    then sealed together with drying cartridges in coated aluminum bags    for long-term storage)-   during the blocking, in an untreated 96-well U-shaped microtiter    plate (polystyrene, from Renner, Order No. 12058), 10 samples+2    positive controls+1 negative control are diluted 1:2 in 1% casein in    PBS, pH 7.2, in 8 stages (starting from 150 μl of sample, 75 μl of    sample are pipetted into 75 μl of casein solution etc.)-   the blocking solution is aspirated out of the microtiter plate    coated with anti-huβ-Gluc antibodies, and 50 μl of the diluted    samples are transferred per well from the dilution plate to the test    plate and incubated at R.T. for 30 min-   during the sample incubation, the ABC-AP reagent (from Vectastain,    Order No. AK-5000) is made up: thoroughly mix 2 drops of reagent A    (Avidin DH) in 10 ml of 1% casein in PBS, pH 7.2, add 2 drops of    reagent B (biotinylated alkaline phosphatase) add mix thoroughly.    (The ABC-AP solution must be made up at least 30′ before use.)-   the test plate is washed 3 times with ELISA washing buffer    (Behringwerke, Order No. OSEW 96)-   50 μl of biotin-labeled detecting antibody mixture (1+1 mixture of    mouse anti 431/26 antibody (MAb 2064/353, Behringwerke) and mouse    anti-CEA antibody (MAb 250/183, Behringwerke) in a concentration of    5 μg/ml diluted in 1% casein in PBS, pH 7.2, final concentration of    each antibody of 2.5 μg/ml) are placed in each well-   the test plate is washed 3 times with ELISA washing buffer-   50 μl of the prepared ABC-AP solution are placed in each well and    incubated at R.T. for 30 min-   during the ABC-AP incubation, the substrate is made up (fresh    substrate for each test: 1 mM 4-methylum-belliferyl phosphate, Order    No. M-8883, from Sigma, in 0.5 M tris+0.01% MgCl₂, pH 9.6)-   the test plate is washed 7 times with ELISA washing buffer-   50 μl of substrate are loaded into each well, and the test plate is    covered and incubated at 37° C. for 2 h-   150 μl of stop solution (0.2 M glycine+0.2% SDS, pH 11.7) are    subsequently added to each well-   the fluorometric evaluation is carried out in a Fluoroscan II (ICN    Biomedicals, Cat. No. 78-611-00) with an excitation wavelength of    355 nm and an emission wavelength of 460 nm-   the unknown concentration of fusion protein in the sample is    determined on the basis of the fluorescence values for the positive    control included in the identical experiment (dilution series with    purified sFv-huβ-Gluc mixed with CEA 5 μg/ml as calibration plot).

EXAMPLE 9

Expression of the sFv-huβ-Gluc Fusion Protein in Yeast.

The single-chain Fv (sFv) from Example 2 is amplified with the oligos2577 (SEQ ID NO:8) and 2561 (SEQ ID NO:7) (FIG. 8) and cloned into thevector pUC19 which has been digested with XbaI/HindIII (FIG. 9).

The human β-glucuronidase gene is amplified with the oligos 2562 (SEQ IDNO:9) and 2540 (SEQ ID NO:10) (FIG. 8) from the plasmid pAB 431/26V_(H)hum/CH1+1H/β-Gluc (Example 3) and ligated into the plasmid sFv431/26 in pUC19 (FIG. 9) cut with BglII/HindIII (FIG. 10).

A KpnI/NcoI fragment is amplified with the oligos 2587 (SEQ ID NO:11)and 2627 (SEQ ID NO:12) (FIG. 8) from the sFv 431/26 and cloned into theyeast expression vector pIXY digested with KpnI/NcoI (FIG. 11).

The BstEII/HindIII fragment from the plasmid sFv 431/26 huβ-Gluc inpUC19 (FIG. 10) is ligated into the vector PIXY 120 which harbors theV_(H) gene, the linker and a part of the V_(L) gene (V_(H)/link/V_(K)part in pIXY 120) and has been digested with BstEII/partially withHindIII (FIG. 12).

The resulting plasmid sFv 431/26 huβ-Gluc in PIXY 120 is transformedinto Saccharomyces cerevisiae and the fusion protein is expressed.

EXAMPLE 10

Expression of the sFv-E. coli-β-glucuronidase Fusion Protein in Yeast.

The E. coli glucuronidase gene is amplified from PRAJ 275 (Jefferson etal. Proc. Natl. Acad. Sci, USA, 83: 8447-8451, 1986) with the oligos2638 (SEQ ID NO:14) and 2639 (SEQ ID NO:13) (FIG. 13) and ligated intosFv 431/26 in pUC19 (Example 9, (FIG. 9) cut with BgIII/HindIII (FIG.14).

A BstEII/HindIII fragment from sFv 431/26 E. coli β-Gluc in pUC19 iscloned into the vector V_(H)/link/V_(K) part in PIXY 120 (Example 9,FIG. 11) which has been partially digested with BstEII/HindIII (FIG.15).

The plasmid sFv 431/26 E. coli β-Gluc in PIXY 120 is transformed intoSaccharomyces cerevisiae and the fusion protein is expressed.

EXAMPLE 11

Expression of the sFv-β-lactamase Fusion Protein in Yeast.

The single-chain Fv (sFv) from Example 2 is amplified with the oligos2587 (SEQ ID NO:15) and 2669 (SEQ ID NO:16) (FIG. 16) and ligated intothe pUC19 vector digested with KpnI/HindIII (FIG. 17).

The β-lactamase II gene (Hussain et al., J. Bacteriol. 164: 223-229,1985) is amplified with the oligos 2673 (SEQ ID NO:17) and 2674 (SEQ IDNO:18) (FIG. 16) from the complete DNA of Bacillus cereus and ligatedinto the pUC19 vector digested with EcoRI/HindIII (FIG. 18). ABclI/HindIII fragment of the β-lactamase gene is ligated into sFv 431/26in pUC19 which has been cut with Bgl II/HindIII (FIG. 19).

The KpnI/HindIII sFv-β-lactamase fragment is ligated into pIXY 120 whichhas been digested with KpnI/partially with HindIII (FIG. 20). Theplasmid is transformed into Saccharomyces cerevisiae, and a fusionprotein which has both the antigen-binding properties of MAb 431/26 andthe enzymatic activity of Bacillus cereus β-lactamase is expressed.

TABLE 1 Pharmacokinetics of sFv-hu β Gluc fusion protein in CD1 nu/numice carrying MzStol ng of sFv-huβGluc per gram of tissue or ml ofplasma measured in the triple determinant test Mouse Mouse Mouse MouseMouse Mouse Tissue 1 2 3 4 5a 5b type 0.05 h 3 h 24 h 48 h 120 h 120 hTumor 24.8 4 7.7 2.1 2.2 6.2 Spleen 15.4 4.1 <0.1 <0.1 <0.1 <0.1 Liver40.9 10.1 0.8 0.8 0.3 <0.1 Intestine 5.2 4.4 1.1 1.2 0.6 <0.1 Kidney44.4 7 <0.1 <0.1 <0.1 <0.1 Lung 154.8 17.3 <0.1 <0.1 <0.1 <0.1 Heart148.3 8.2 <0.1 <0.1 <0.1 <0.1 Plasma 630.9 95 2.7 0.4 <0.1 <0.1 i.v.injection of 0.8 μg of purified fusion protein per mouse

TABLE 2 Analysis of the monosaccharide components in the carbohydratecontent of the sFv-huβ-Gluc fusion protein from BHK cells The purifiedsFv-huβ-Gluc fusion protein was investigated for its carbohydratecontent. This revealed after hydrolysis the following individualcomponents in the stated molar ratio (mol of carbohydrate/mol ofsFv-huβ-Gluc). N-Acetyl N-Acetyl- Fucose Galactosamine glucosamineGalactose Glucose Mannose neuraminic acid sFv-huβ-Gluc 4 2 30 8 1 43 4

The molar ratios of mannose, glucosamine and galactose allow conclusionsto be drawn about the presence of the high-mannose type and/or hybridstrutures (besides complex type structyres). Therefore mannose,galactose, acetylneuraminic acid and possibly acid and possiblyN-acetylglucosamine occur terminally, and mannose, may also be presentas mannose 6-phosphate.

Methods:

Neuraminic acid was determined by the method of Hermentin and Seidat(1991) GBF Monographs Volume 15, pp. 185-188 (after hydrolysis for 30min in the presence of 0.1 N sulfuric acid at 80° C. and subsequentneutralization with 0.4 N sodium hydroxide solution) by high-pH anionexchange chomatography with pulsed amerometric detection (HPAE-PAD).

The monosaccaride components were determined (after hydrolysis for 4 hin the presence of 2 N trifluoracetic acid at 100° C. and evaporation todryness in a SpeedVac) likewise by HPAE-PAD in a motivation of themethod described by Hardy et al. (1988) Analytical Biochemistry 170, pp.54-62.

TABLE 3 Analysis of the monosaccharide components in the carbohydratecontent of the sFv-huβGluc fusion protein from Saccharomyces cerevisiae.Glucosamine Glucose Mannose sFv-huβGluc 6 12 150 mol/mol (mol/mol)

1. A eukaryotic host cell comprising a nucleic acid sequence coding fora compound comprising two or more antigen bindig regions linked to atleast one prodrug-activating enzyme, wherein a) the antigen bindingregion consists of a single polypeptide chain; b) the single polypeptidechain is comprised of a first variable domain, a second variable domainand a polypeptide linker connection the first variable donmain and thesecond variable domain, wherein the nucleotide sequence the polypeptidelinker is formed by two partially overlapping PCR primers during a PCRreaction that links the first variable domain and the second variabledomain; and wherein c) said compound has a bivalent or a multivalentstructure and is glycosylated.
 2. The host cell of claim 1 wherein thehost cell contains an expression vector comprising the nucleic acidsequence.
 3. The host cell of claim 1, wherein sajd host cell isselected from the group consisting of BHK, CHO, COS, Hela, insect,tobacco plant, and yeast cell.
 4. A process for preparing a compoundwhich comprises cultivating the host cell of claim 1 and isolating saidcompound.
 5. A transgenic mammal that is not human comprising a nucleicacid sequence coding for a compound comprisding two or more antigenbinding regions linked to at least one prodrug-activating enzyime,wherein a) the antigen binding region consists of a single polypeptidechain; b) the single polypeptide chain is comprised of a first variabledomain, a second variable domain and a polypeptide linker connecting thefirst variable domain and the second variabi domain, wherein thenucleotide sequence encoding the polypeptide linker is formed by twopartially overlapping PCR primers during a PCR reaction that links thefirst variable domain and the second variable domain; and wherein c)said compound has a bivalent or a multivalent structure and isglycosylated.
 6. The eukaryotic host cell of claim 1 or the transgenicmammal of claim 5 wherein at least one antigen binding region comprisesa variable domain of a heavy antibody chain and a variable domain of alight antibody chain (sFv fragment).
 7. The eukaryotic host cell ofclaim 1 or the transgenic mammal of claim 5 wherein at least one of theantigen binding regions binds to a tumor-associated antigen (TAA). 8.The eukaryotic host cell or the transgenic mammal claim 7, wherein saidTAA is selected from the group consisting of an N-CAM, PEM, EGF-R,Sialyl-Le^(a), Sialyl-Le^(x), TFβ, GICA, GD₃, GD₂, TAG72, CA125, the24-25 kDa glycoprotein defined by MAb L6 and CEA.
 9. The eukaryotic hostcell of claim 1 or the transgenic mammal of claim 5 wherein saidprodrug-activating enzyme is selected from the group consisting of alactamase, pyroglutamate aminopeptidase, D-aminopeptidase, oxidase,peroxidase, phosphatase, hydroxylnitrile lysase, protease, esterase,carboxypeptidase and glycosidase.
 10. The eukaryotic host cell or thetransgenic mammal claim 9, wherein the enzyme is a β-glucuronidase,which is selected from the group consisting of an E. coilβ-glucuronidase, a Kobayasia nipponica β-glucuronidase, a Secale cerealeβ-glucuronidase and a human β-glucuronidase.
 11. The eukaryotic hostcell of claim 1 or the transgenic mammal of claim 5, wherein at leastone of the antigen binding regions is linked to the enzyme via a peptidelinker.
 12. The eukaryotic host cell of claim 1 or the transgenic mammalof claim 5 wherein said nucleic acid sequence codes for a humanized sFvfragment against CEA and a human β-glucuronidase.
 13. The eukaryotichost cell or the transgenic mammal of claim 12, wherein said nucleicacid sequence is SEQ ID NO:
 1. 14. The eukaryotic host cell of claim 1or the transgenic mammal of claim 5 wherein said nucleic acid sequenceencodes SEQ ID NO:2.